The goal of this proposal is to determine the mechanism and specificity of human DNA ligases. All organisms have an absolute requirement for DNA replication and DNA repair in order to synthesize new cells and to maintain correct cellular functions. DNA ligases catalyze the ultimate step in DNA replication and most DNA repair pathways, however most research has focused on DNA polymerases and therefore we lack a fundamental understanding of DNA ligase mechanisms and specificities. It is essential to learn the molecular mechanism of human DNA ligases and that these insights will have significant value for understanding human health. Our work is guided by the known biology of DNA repair and replication and we focus on unanswered puzzles that cannot be explained by current understanding of these pathways. Some recent clinical examples of where this knowledge is critical is in patients with inherited mutations in DNA ligase genes, such as LIG1 syndrome which causes a Primary ImmunoDeficiency and LIG4 syndrome which causes Severe Combined ImmunoDeficiency (SCID), and in abnormal states such as cancer in which ligases, especially LIG3, have been overexpressed. We will use quantitative mechanistic enzymology and x-ray crystallography (in collaboration with Dr. Scott Williams) to characterize human DNA ligase 1 (LIG1) and DNA ligase 3 (LIG3). Genetic observations suggest that these enzymes have unique functions, but share some similarities. For example, LIG1 and LIG3 both function in DNA repair and replication in the nucleus, but only LIG3 functions in the mitochondria. We will extend our previous kinetic and structural studies of LIG1 to understand specificity of this enzyme and to define the minimal steps in locating and engaging a single strand break. We have recently succeeded in producing large quantities of recombinant LIG3 alpha and beta isoforms and we will perform a kinetic and thermodynamic characterization to understand similarities and differences with LIG1. For both enzymes, we will use site-directed mutagenesis to target specific functions, such as metal cofactor binding, DNA binding, and catalytic specificity. Analysis of these mutant proteins will provide an understanding of the molecular features of eukaryotic DNA ligation that will be invaluable to understand ligase function in normal cells and in human disease. The core objectives of the grant are to develop a molecular understanding of the mechanism and specificity of LIG1 and LIG3, but the mechanistic models that emerge will then be tested in an appropriate cell. This work will uncover the interconnections between different DNA repair pathways and provide a strong foundation for understanding regulatory interactions and modifications.